Prevalence of Trypanosoma and Sodalis in wild populations of tsetse flies and their impact on sterile insect technique programmes for tsetse eradication

The sterile insect technique (SIT) is an environment friendly and sustainable method to manage insect pests of economic importance through successive releases of sterile irradiated males of the targeted species to a defined area. A mating of a sterile male with a virgin wild female will result in no offspring, and ultimately lead to the suppression or eradication of the targeted population. Tsetse flies, vectors of African Trypanosoma, have a highly regulated and defined microbial fauna composed of three bacterial symbionts that may have a role to play in the establishment of Trypanosoma infections in the flies and hence, may influence the vectorial competence of the released sterile males. Sodalis bacteria seem to interact with Trypanosoma infection in tsetse flies. Field-caught tsetse flies of ten different taxa and from 15 countries were screened using PCR to detect the presence of Sodalis and Trypanosoma species and analyse their interaction. The results indicate that the prevalence of Sodalis and Trypanosoma varied with country and tsetse species. Trypanosome prevalence was higher in east, central and southern African countries than in west African countries. Tsetse fly infection rates with Trypanosoma vivax and T. brucei sspp were higher in west African countries, whereas tsetse infection with T. congolense and T. simiae, T. simiae (tsavo) and T. godfreyi were higher in east, central and south African countries. Sodalis prevalence was high in Glossina morsitans morsitans and G. pallidipes but absent in G. tachinoides. Double and triple infections with Trypanosoma taxa and coinfection of Sodalis and Trypanosoma were rarely observed but it occurs in some taxa and locations. A significant Chi square value (< 0.05) seems to suggest that Sodalis and Trypanosoma infection correlate in G. palpalis gambiensis, G. pallidipes and G. medicorum. Trypanosoma infection seemed significantly associated with an increased density of Sodalis in wild G. m. morsitans and G. pallidipes flies, however, there was no significant impact of Sodalis infection on trypanosome density.

www.nature.com/scientificreports/ found in all laboratory-reared tsetse colonies and some wild populations 21 indicates that mitigating action, such as feeding the flies 2-3 times on blood supplemented with trypanocidal drugs before release, is required in SIT programs to minimize the risk of disease transmission by the large number of released males that harbour Sodalis. Field studies in two HAT foci in Cameroon used PCR to detect Trypanosoma and Sodalis in G. palpalis palpalis and the results indicate that the presence of Sodalis favours Trypanosoma infections especially by T. brucei s.l. 30 . Furthermore, in the wildlife-livestock-human interface in the Maasai Mara National Reserve in Kenya, it was shown that G. pallidipes infected with Sodalis was associated with increased Trypanosoma infection rates 31 . However, other studies have found no strong association between trypanosome and Sodalis in some tsetse species collected in four locations in Kenya 32 . Channumsin et al., 33 suggested that the association between Trypanosoma infection and the presence of Sodalis will vary depending on tsetse and Trypanosoma species. Similarly, studies carried out in the Fontem focus in Cameroon did not find a relationship between the endosymbiont and the parasite in G. p. palpalis 34 , and no significant Sodalis-Trypanosoma infection association was found in G. tachinoides in two sites of the Faro and Déo Division in Adamawa region of Cameroon 35 . Likewise, no association between the presence of the parasite and Sodalis was found in G. brevipalpis, G. m. morsitans and G. pallidipes in the Luambe National Park of Zambia 36 .
The overall objective of this study was to evaluate the prevalence of Sodalis and Trypanosoma in wild tsetse populations at a continental scale, i.e. Burkina Faso, Democratic Republic of Congo (DRC), Eswatini, Ethiopia, Ghana, Guinea, Kenya, Mali, Mozambique, Senegal, South Africa, Tanzania, Uganda, Zambia, and Zimbabwe and analyse these data in the context of a possible association between the occurrence of Sodalis and Trypanosoma infection in tsetse. Such information might guide the decision maker for SIT programmes to take the appropriate action, if necessary, to minimize any potential risk of increased transmission.

Results
Trypanosoma prevalence. Adult  The results indicate that 1736 (25.3%) adults were infected with one or more Trypanosoma taxa (Tables 1, 2 and 3), The Trypanosoma prevalence varied significantly between tsetse taxa (X 2 = 750.18, df = 9, P << 0.001) and between countries (X 2 = 2038.1, df = 14, P << 0.001). The Permanova analysis indicated as well significant differences between countries (P = 0.009) and taxa (P = 0.041) ( Table 4). As all taxa were not collected from all countries, the interaction between taxa and countries was only analyzed where a taxon was collected from several countries.
Regardless of tsetse taxon, in west African countries the average Trypanosoma prevalence was 20% (n = 3733), with the highest prevalence recorded in Ghana (61%) and the lowest recorded in Guinea (2.2%). The prevalence in Burkina Faso, Mali and Senegal was 21.9, 6.9 and 14.2% respectively (Fig. 1, and Table 1). In east, central and southern African countries, the Trypanosoma infection prevalence was a bit higher than in west African countries with an averaged infection of 31.5% (n = 3127), with the highest prevalence (53.6%) in Zimbabwe and lowest prevalence (2.9%) in DRC. No Trypanosoma infection was detected in Eswatini ( Fig. 1 and Table 1). Regardless of the country, Trypanosoma prevalence varied from one taxon to another, and G. m. morsitans showed the highest Trypanosoma prevalence (41%) followed by G. pallidipes (38.5%) and the lowest prevalence was detected Table 1. Global prevalence of Sodalis and Trypanosomes in tsetse samples analyzed per country. *Values indicated by the same lower-case letter do not differ significantly at the 5% level.  Table 2). Some tsetse taxa were collected from several countries as presented in Fig. 2 and Table 3. The highest Trypanosoma prevalence was recorded in G. tachinoides in Ghana (61%). This was followed by high prevalence in G. m. morsitans collected from Zimbabwe (53.9%), Tanzania (53%) and Zambia (48.4%). G. pallidipes from Zimbabwe, Kenya, Zambia and Tanzania also showed high Trypanosoma prevalence of 52.7%, 50.9%, 45.2% and 37.3%, respectively. The lowest Trypanosoma prevalence was found in G. p. gambiensis from Guinea (2.2%). Based on the Trypanosoma prevalence presented in Fig. 2 and Table 3, the tested samples can be categorized as: (i) tsetse samples with high prevalence (> 35%) detected in G. tachinoides from Ghana; G. medicorum from Burkina Faso,   www.nature.com/scientificreports/ prevalence for each tsetse species, the differences were significant only in G. p. gambiensis (X 2 = 26.71, df = 4, P < 0.001) and G. tachinoides, (X 2 = 9.38, df = 1, 2, P = 0.002). In contrast, no significant difference was detected between countries for G. austeni (X 2 = 1.47, df = 4, P = 0.688), G. brevipalpis (X 2 = 0.34, df = 2, P = 0.559), G. f. fuscipes (X 2 = 0.15, df = 2, P = 0.702), G. m. morsitans (X 2 = 1.04, df = 3, P = 0.593) and G. pallidipes (X 2 = 4.983, df = 1,6, P = 0.418) (  The prevalence of the different Trypanosoma species with respect to the above-mentioned groups, indicate that infections with the Tsg group was the highest regardless of countries or tsetse species with an average of 7.06%. The infection rate was higher (14.13%) in east, central and southern African countries than in west Africa (1.13%). Tv infection averaged at 6.75% but with higher prevalence in west African countries (10.37%) than in east, central and southern Africa (2.43%). The prevalence of Tc infection was lower than Tv and Tsg group with an average of 4.78% with higher prevalence in central and southern Africa (8.38%) than in west Africa (1.77%). The Tz group had the lowest prevalence with an average of 2.29%. Like Tv infection, the Tz prevalence was higher in west Africa (3.16%) than central and southern Africa (1.25%).
Mixed infections of Trypanosoma groups (double or triple) are rare events with an average prevalence between 0.09 and 1.71% regardless of country or tsetse species. However, double infections seem to be more frequent in some countries than others (X 2 = 35.01, df = 14, P = 0.001) for Tv-Tz and in some tsetse species than others (X 2 = 21.20, df = 9, P = 0.012) for Tv-Tz (Supplementary File 1). The highest prevalence of the mixed infections Tv-Tz and Tc-Tz were observed in Ghana with 12.39% and 10.68%, respectively, regardless of tsetse species. Although the average Tc-Tsg prevalence was higher than that of Tv-Tz and Tc-Tz, the highest mixed infection with it was found in Zambia with 9.05%. Regardless of the country, the highest mixed infection of Tc-Tsg detected per tsetse species was ~ 5% in G. m. morsitans and G. pallidipes. The mixed infection of Tsg with either Tv or Tz or both was lower than 2% regardless of the country or tsetse species. Taking into account both the country and tsetse species, the highest mixed infection of Tc-Tsg (12.5%) was detected in G. m. morsitans in Zambia. However, the highest prevalence of Tc-Tz (10.68%) and Tv-Tz (12.39%) was detected in G. tachinoides from Ghana. Although the average prevalence of Tv-Tsg was low (0.54%), a relative high infection rate of 6.17% was found in G. m. morsitans from Tanzania.
A triple infection of Trypanosoma groups (Tc-Tv-Tz) was only detected in G. medicorum from Burkina Faso (1.30%) and G. tachinoides from Ghana (1.71%) ( Fig. 2  Prevalence of Sodalis infection. The prevalence of Sodalis infection based on the PCR results varied significantly with country (X 2 = 108.02, df = 1, 14, P << 0.001) and tsetse species (X 2 = 69.60, df = 9, P < 0.001). The best glm model (lowest AICc) selected for the overall Sodalis prevalence retained the countries, the species and their interaction (where possible) as variables that fitted the data well (AICc = 1296.12). Similar to the prevalence of Trypanosoma, the average Sodalis prevalence in east, central and southern Africa (24.6%) was higher than in west Africa (2.70%). Regardless of tsetse species, the highest prevalence of Sodalis infection was found in Tanzania (67.1%) followed by Uganda (43.3%), Kenya (28.5%) and Ethiopia (20.48%) (  www.nature.com/scientificreports/ taxon showed significant deviation from independence at the Bonferroni corrected α = 0.00833 in G. pallidipes (P < 0.001) and G. p. gambiensis (P < 0.001) (Supplementary Table 4). The prevalence of coinfection of Sodalis and Trypanosoma in wild tsetse populations varied with tsetse taxon and location. No coinfection was found in many taxa and many locations. The co-infection was found only in G. f. fuscipes (2.73%), G. m. morsitans (15.72%) and G. pallidipes (9.22%) in east, central and southern Africa (Fig. 3B, Table 5 and Supplementary Table 4).

Impact of co-infection on Trypanosoma and Sodalis density.
Attempts were to assess the density of Trypanosoma and Sodalis under single (S − /T + ) and (S + /T − ) or double infection (S + /T + ) conducted using qPCR with primers mentioned in Supplementary Table 5. The results show that Sodalis infections did not have a significant impact on Trypanosoma density (X 2 = 0.648, df = 2, P = 0.723), however the median value of (S + /T + ) files were slightly (S − /T + ) lower than (S + /T − ) and (S − /T + ) flies and the number of outlier samples with higher trypanosome density (S − /T + ) flies (Fig. 4A). Trypanosoma infections significantly reduced the density of Sodalis as indicated by comparing (S + /T + ) flies with (S + /T − ) flies (P = 0.014) although the median values in (S + /T + ) files is higher than the other samples indicating that the increased of Sodalis density in (S + /T − ) might be affected with the outlier flies with high Sodalis density (Fig. 4B). No significant different was found in the Trypanosoma density determined by qPCR in the flies tested negative (S + /T − ) or positive (S + /T + ) and (S − /T + ) with the standard PCR, however, Sodalis density showed significant difference between flies with different infection type (X 2 = 14.54, df = 2, P < 0.001) (Fig. 4B). The results showed no correlation between Sodalis and Trypanosoma density (r = 0.007, t = 0.055, df = 69, P = 0.9561) Supplementary Fig. 2, Supplementary File 1).

Discussion
The implementation of the SIT in the context of an AW-IPM strategy to eradicate tsetse flies relies on the release of sterile males in the targeted area. This was successful in eradicating a population of G. austeni from Unguja Island of Zanzibar 37 and significant progress has been made in the eradication programme implemented against G. p. gambiensis in the Niayes area of Senegal 38 . However, as both male and female tsetse flies are vectors of Trypanosoma species, the release of large numbers of sterile male flies bears a potential risk of temporarily increasing disease transmission during the initial release phase of an SIT programme 39 . Therefore, mitigating measures are required to reduce or eliminate this potential risk, especially in areas where sleeping sickness (HAT) is endemic.
To date, to mitigate such risks, sterile males are offered two or three blood meals mixed with the trypanocidal drug isometamidium chloride, before being released which reduces the risk of Trypanosoma transmission significantly but does not eliminate it 40,41 . In addition, other approaches were proposed to minimize such risks such as paratransgenesis 42,43 and combining paratransgenesis with SIT 44 . The vector competence of tsetse flies for different trypanosome species is highly variable and is suggested to be affected by various factors, amongst which bacterial endosymbionts. Here, the interaction of Sodalis glossinidius with tsetse trypanosome infection is still under debate. Several studies reported a potential positive correlation between Sodalis and Trypanosoma infections 28,30,32,36,[45][46][47][48] , leading to the hypothesis that Sodalis might facilitate the establishment of Trypanosoma infections in the tsetse midgut 23,26,27 . However, other studies indicated the lack of correlation between Sodalis and Trypanosoma infection [34][35][36] . The presence of Sodalis infections in tsetse rearing colonies has been well studied and previous studies indicated that Sodalis is more frequently present in colonized tsetse flies than in wild tsetse populations 36,49 with a prevalence of 80 and 100% in colonized G. m. morsitans and G. p. gambiensis, respectively 49,50 , which is higher than the symbiont prevalence in wild populations of these tsetse species. This seems to indicate that the rearing process of tsetse flies favours the transmission and spread of Sodalis infections within the colonized population. Recently, colonies of G. pallidipes, G. p. gambiensis, G. f. fuscipes, G. m. morsitans, G. m. centralis and G. m. submorsitans maintained at the FAO/IAEA Insect Pest Control Laboratory were screened for Sodalis infections and showed a 100% prevalence of Sodalis; only the G. brevipalpis colony had a lower prevalence of 95% (data not shown). Taken into consideration that mass-rearing conditions enhances Sodalis infections and that Sodalis infections might facilitate the establishment of a Trypanosoma infection in the midgut, sterile male tsetse flies that are derived from colonies might be effective vectors for different Trypanosoma species and, therefore, might increase the trypanosome transmission after flies being released. It is therefore important that the managers and planners of SIT programmes are aware which tsetse species show a positive correlation between Sodalis and Trypanosoma infections to be able to take the necessary mitigating actions.
Various studies have examined the prevalence of Sodalis and Trypanosoma species in wild tsetse populations 30,32,35,45,51 , but our study presents for the first time the prevalence of Sodalis and Trypanosoma species on a continent-wide scale. In addition, the DNA extraction and PCR methods we have used were standardized and were all carried out in one laboratory to avoid discrepancies in the results due to different handling of tsetse samples or to different methods to discriminate trypanosome species in tsetse tissues. Our results indicate that Sodalis and Trypanosoma prevalence varied with tsetse species and geographical location (with an overall trypanosome prevalence of 23.5%), which agrees with many previous studies 52 . A high Trypanosoma prevalence (> 30%) was found in G. m. morsitans and G. pallidipes from central and east Africa. This finding is in agreement with previous reports on G. m. morsitans and G. m. centralis from Zambia 36,52 and G. m. morsitans sampled in Malawi 53 . Moreover, a high prevalence of Trypanosoma infection in G. pallidipes was also previously reported in Tanzania 54 and Kenya 33 . However, another study in northern Tanzania indicated a lower prevalence of Trypanosoma infection (< 10%) both in G. m. morsitans and G. pallidipes 55 .
Our study showed that the prevalence of different Trypanosoma species and or subspecies can be different in different tsetse taxa. In G. tachinoides in Ghana, the Trypanosoma vivax (Tv) infection was high (> 10%) as well as the infections of the T. brucei sspp (Tz) and the T. simiae/T. godfreyi (Tsg) group and the mixed infections of Tv-Tsg. In contrast, the prevalence of T. congolense was very low. These results are in agreement with the www.nature.com/scientificreports/ prevalence of T. brucei s.l (11%) and T. congolense forest type (2.6%) reported in the same tsetse species in Cameroon. The same study reported a prevalence of 13.7% of T. congolense savannah type 35 , which was not observed in our study. Our results of trypanosome infection rates in G. tachinoides also agree with former studies 56,57 , except for T,c for which a high fly infection rate (31.8%) was previously shown 57 . The Tc infection rates in our study were high in G. pallidipes and G. m. morsitans; for the latter tsetse fly species, a study in Malawi reported a high prevalence for T. brucei (64.4%) but much lower for all other Trypanosoma infections(< 10%) 58 . The mixed infection of Trypanosoma species/subspecies is in agreement with previous reports 35,52,57,59 . Likewise, the prevalence of Sodalis infection varied significantly with tsetse taxon and location and the highest prevalence was found in G. m. morsitans and G. pallidipes. Our results agree with the high prevalence of Sodalis reported in G. pallidipes (~ 50%) in one location in Kenya regardless of the fly age 33 ; however, the same study reported low Sodalis prevalence in another location. In another study in Kenya, Wamwiri et al., 32 reported moderate Sodalis prevalence in G. pallidipes (16%) and low prevalence in G. austeni (3.7%), which is in agreement with our results. On the other hand, our results are different from the low prevalence (< 8%) found in G. m. morsitans and G. pallidipes in Zambia 36 . In another study in Zambia, Sodalis prevalence in G. m. centralis, was reported to be 15.9% with no significant difference between inter-site prevalence 52 . The prevalence of Sodalis in G. brevipalpis in our study was found to be low (< 2.3%) which contradicts with the high prevalence (93.7%) reported in this species in Zambia 36 . In the DRC, the global prevalence of Sodalis in G. fuscipes quanzensis midgut averaged 15.5%, but in certain locations the prevalence exceeded 40% 60 . In Nigeria, Sodalis prevalence in G. p. palpalis and G. tachinoides was 35.7% 61 which is higher that the prevalence reported in our study for both species.
Our data indicate that the Trypanosoma and Sodalis infections were very low or absent in some tsetse taxa from certain locations such as G. austeni in Eswatini for Trypanosoma and Sodalis infections and several species in west Africa for Sodalis. The lack of Sodalis and/or Trypanosoma infection in these samples might be due to (i) low number of tested samples (ii) the use of the DNA extracted from the whole body of tsetse adults (iii) the possibility of the collected samples being infected with different strains/genotypes that might not be detected with the primers used and (iv) the infection of Sodalis and Trypanosoma are under the detection limit of the used PCR. It is important to note that due to the high number of samples tested in our study, the more sensitive nested PCR to detect low infection level was excluded due to logistic reasons.
Our results indicate significant deviation from independence (correlation) of Sodalis and Trypanosoma infections in G. medicorum, G. p. gambiensis and G. pallidipes. However, the lack of detection of any tsetse adult with co-infection of Sodalis and Trypanosoma in G. medicorum, and G. p. gambiensis might indicate a negative correlation. Such negative trend might be supported by the lower density of Sodalis in the flies with co-infection (S + /T + ) compared to these with Sodalis infection only (S + /T − ). On other hand the lack of impact of Sodalis infection on Trypanosoma density does not support the negative trend and agreed with the results of Trappeniers et al., 24 reported on colonized flies. This results also agreed with previous results reporting the absence of direct correlation between the presence of Sodalis and the acquisition of a Trypanosoma infection 63 . However, an inverse correlation was reported between Sodalis and the vector competence where the presence of Sodalis in both midgut and proboscis of G. p. gambiensis was associated with its status as a poor vector, whereas it is not found in the proboscis of G. m. morsitans (major vector). It is worth noting that all previous studies of Sodalis infection in G. p. gambiensis and its interaction with Trypanosoma infection was carried out with flies reared under laboratory conditions 28,29,64 . The correlation between Sodalis and Trypanosoma infection in G. pallidipes is   www.nature.com/scientificreports/ positive, evidenced with the relative high number (n = 170) of tsetse with co-infection. This positive correlation was also found in G. pallidipes from Kenya although with too few flies with co-infection to enable us to draw a definite conclusion 32 . Although co-infections were found in G. m. morsitans and G. f. fuscipes in some locations, the global correlation was missing. This is in agreement with the positive correlation found between Sodalis and Trypanosoma infection in G. m. centralis in Zambia, in which there was a 6.2 fold increase in the likelihood of a fly being infected with Trypanosoma if Sodalis was present 52 . More studies are needed to enhance the potential control interventions mediated by endosymbionts to reduce parasitic infections 61 .
The results of this study clearly indicate that the interaction between Sodalis and Trypanosoma infection is complex, species-specific and requires further investigation. The prevalence results indicate that Sodalis and Trypanosoma infections are not independent in some species, such as G. p. gambiensis and G. medicorum in west Africa and G. pallidipes in central and east Africa, In case of a positive correlation between Sodalis and Trypanosoma infection in these species, additional measures could be suggested when implementing the SIT to reduce the Sodalis density in the sterile males released in the targeted area to maximize the safe implementation of the SIT. These measures might include the mixing of Sodalis phage(s) 29,65 with the blood meals to feed the mass-reared flies to reduce the Sodalis density in these flies. In addition, the blood meal offered to the males before release can be supplemented with one or more of the following antimicrobial products to reduce Sodalis density, i.e. streptozotocin 23 , indolicidin and OaBAC 5 mini 66 . The use of the Sodalis phage as well as these antimicrobial agents requires further studies to (1) develop methods to isolate the phage, (2) determine the conditions (e.g. suitable concentration) for its use, and (3) determine the impact on Sodalis density, tsetse productivity and

Conclusion
Sodalis and Trypanosoma infection varied with tsetse taxon and location. There is a significant positive correlation between Sodalis and Trypanosoma infection in G. medicorum, G. p. gambiensis and G. pallidipes; however, no significant correlation was found in other tsetse taxa and locations. The results of this study will enable the decision makers of SIT projects to better plan and take the necessary measures to fine-tune and optimize SIT efficiency and safety.   Table 5) and only the successful samples were included in the analysis 21,75 .

Methods
Trypanosoma prevalence and genotyping. Polymerase chain reaction (PCR), following the method of Njiru et al. 76 that used the primers ITS1-CF and ITS1-BR (Supplementary Table 5 Table 5) to assess the density of the symbiont present within Trypanosoma infected and noninfected, additional criteria for the selection of the samples was the presence of the two groups (S + /T + ) and (S + /T − ) in a given population. Based on the preceding criteria 96 individual flies (52 and 44 flies with infection status of (S + /T + ) and (S + /T − ), respectively, were selected from the G. pallidipes and G. m. morsitans collected in Kenya, Tanzania and Zimbabwe. In addition, samples with (S + /T + ) and (S − /T + ) were used to assess the impact of Sodalis infection on Trypanosoma density. Trypanosomatidae18S specific primers (18S_Typ_F and18S_Typ_R) (Supplementary Table 5) were used to assess the Trypanosoma density in the tested samples. The DNA from all selected samples was diluted to a final concentration of 4 ng/μl and 5 μl of the diluted DNA was used for qPCR to determine Sodalis and Trypanosoma DNA density normalized to the housekeeping β-tubulin gene. The amplification mixture contained 5 μl of DNA template, 200 nM of each primer, and 7.5 μl iQ™ SYBER Green Supermix (Bio-Rad). qPCR cycling conditions for Sodalis were as follows: initial denaturation at 95 °C for 2 min; 39 cycles of 95 °C for 5 s, 55 °C for 30 s, one step at 95 °C for 5 s and a melting curve constructed from 65 °C to 95 °C in increments of 0.5 °C for 5 s. The same conditions were used for Trypanosoma except the annealing temperature was at 60 °C. The analysis of the Sodalis, Trypanosoma and Tubulin densities was based only on qPCR data with the expected melting curve at 81.5 °C, 85.5 °C and 86 °C, respectively. Data analysis. The prevalence data were recorded and analyzed with the general linear model (GLM) 79 .
The prevalence of Sodalis, Trypanosoma species and each Trypanosoma species and co-infection were tested for differences between the tsetse taxa and between countries. For each country, the prevalence was assessed again for differences between the localities where the flies were collected and between the tsetse species present in each country. In the absence of PCR detected Sodalis or Trypanosoma infection, the upper 95% confidence interval for the true rate of infection was calculated following the method of Couey and Chew 62 . Trypanosoma prevalence between taxa was compared between species by a pairwise comparison of proportions with a Bonferroni correction and Benjamini-Hochberg correction. The analyses were executed in R v 4.0.5 79  To analyse the qPCR data, normalized density of Trypanosoma and Sodalis against the house keeping gene (tubulin) was extracted from the CFX Maestro software. Samples giving a valid density (not N/A) for both Trypanosoma and Sodalis were retained for further statistical analysis in R. Similarities in the structure of Sodalis and Trypanosoma (single and multiple) infection and the role of different factors such as countries and tsetse taxa, were assessed using the matrix display and metric multidimensional scaling (mMDS) plot with bootstrap averages in PRIMER version 7+. The bootstrap averages plots were displayed with a Bray and Curtis matrix based on the square-root transformation of the Sodalis and Trypanosoma (single and multiple) infection abundance data 87 . The tests were based on the multivariate null hypothesis via the use of the non-parametric statistical method PERMANOVA 88 . The Permanova test was conducted on the average of the abundance data based on the country-species after excluding the data of Eswatini (low number of tested samples).